Curvature Footprints of Transmembrane Proteins in Simulations with the Martini Force Field.

Membranes play essential roles in biological systems and are tremendously diverse in the topologies and chemical and elastic properties that define their functions. In many cases, a given membrane may display considerable heterogeneity, with localized clusters of lipids and proteins exhibiting distinct characteristics compared to adjoining regions. These lipid-protein assemblies can span nanometers to micrometers and are associated with cellular processes such as transport and signaling. While lipid-protein assemblages are dynamic, they can be stabilized by coupling between local membrane composition and shape. Due to the inherent difficulty in resolving atomistic details of membrane proteins in their native lipid environments, these complexes are notoriously challenging to study experimentally; however, molecular dynamics (MD) simulations might be a viable alternative. Here, we aim to assess the utility of coarse-grained (CG) MD simulations with the Martini force field for studying membrane curvature induced by transmembrane (TM) proteins that are reported to generate local curvature. The direction and magnitude of curvature induced by five different TM proteins, as well as certain lipid-protein and protein-protein interactions, were found to be in good agreement with available reference data.

Molecular dynamics simulations of lipid-protein interactions in SLC4 proteins.

The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO3-, CO32-, Cl-, Na+, K+, NH3, and H+, which are necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl-/HCO3- exchanger) and NBCe1 (Na+-CO32-cotransporter). Previous computational studies of the outward-facing state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed coarse-grained and atomistic molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1, and NDCBE (an Na+-CO32-/Cl- exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The recently resolved inward-facing state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, phosphatidylcholine, and sphingomyelin in the three studied proteins, and their potential roles in the SLC4 transport function, conformational transition, and protein dimerization are discussed.

The structure, self-assembly and dynamics of lipid nanodiscs revealed by computational approaches.

Nanodisc technology is increasingly being used in structural, biochemical and biophysical studies of membrane proteins. The computational approaches have revealed many important features of nanodisc assembly, structures and dynamics. Therefore, we reviewed the application of computational approaches, especially molecular modeling and molecular dyncamics (MD) simulations, to characterize nanodiscs, including the structural models, assembly and disassembly, protocols for modeling, structural properties and dynamics, and protein-lipid interactions in nanodiscs. More amazing computational studies about nanodiscs are looked forward to in the future.

Unveiling Interactions of Tumor-Targeting Nanoparticles with Lipid Bilayers Using a Titratable Martini Model.

pH-responsive nanoparticles are ideal vehicles for drug delivery and are widely used in cell imaging in targeted therapy of cancer, which usually has a weakly acidic microenvironment. In this work, we constructed a titratable molecular model for nanoparticles grafted with ligands of pH-sensitive carboxylic acids and investigated the interactions between the nanoparticles and the lipid bilayer in varying pH environments. We mainly examined the effect of the grafting density of the pH-sensitive ligands of the nanoparticles on the interactions of the nanoparticles with the lipid bilayer. The results show that the nanoparticles can penetrate the lipid bilayer only when the pH value is lower than a critical value, which can be readily modulated to the specific pH value of the tumor microenvironment by changing the ligand grafting density. This work provides some insights into modulating the interactions between the pH-sensitive nanoparticles and cellular membranes to realize targeted drug delivery to tumors based on their specific pH environment.

Structural characterization of the antimicrobial peptides myxinidin and WMR in bacterial membrane mimetic micelles and bicelles.

Antimicrobial peptides are a promising class of potential antibiotics that interact selectively with negatively charged lipid bilayers. This paper presents the structural characterization of the antimicrobial peptides myxinidin and WMR associated with bacterial membrane mimetic micelles and bicelles by NMR, CD spectroscopy, and molecular dynamics simulations. Both peptides adopt a different conformation in the lipidic environment than in aqueous solution. The location of the peptides in micelles and bicelles has been studied by paramagnetic relaxation enhancement experiments with paramagnetic tagged 5- and 16-doxyl stearic acid (5-/16-SASL). Molecular dynamics simulations of multiple copies of the peptides were used to obtain an atomic level of detail on membrane-peptide and peptide-peptide interactions. Our results highlight an essential role of the negatively charged membrane mimetic in the structural stability of both myxinidin and WMR. The peptides localize predominantly in the membrane's headgroup region and have a noticeable membrane thinning effect on the overall bilayer structure. Myxinidin and WMR show a different tendency to self-aggregate, which is also influenced by the membrane composition (DOPE/DOPG versus DOPE/DOPG/CL) and can be related to the previously observed difference in the ability of the peptides to disrupt different types of model membranes.

Martini 3 Force Field Parameters for Protein Lipidation Post-Translational Modifications.

Protein lipidations are vital co/post-translational modifications that tether lipid tails to specific protein amino acids, allowing them to anchor to biological membranes, switch their subcellular localization, and modulate association with other proteins. Such lipidations are thus crucial for multiple biological processes including signal transduction, protein trafficking, and membrane localization and are implicated in various diseases as well. Examples of lipid-anchored proteins include the Ras family of proteins that undergo farnesylation; actin and gelsolin that are myristoylated; phospholipase D that is palmitoylated; glycosylphosphatidylinositol-anchored proteins; and others. Here, we develop parameters for cysteine-targeting farnesylation, geranylgeranylation, and palmitoylation, as well as glycine-targeting myristoylation for the latest version of the Martini 3 coarse-grained force field. The parameters are developed using the CHARMM36m all-atom force field parameters as reference. The behavior of the coarse-grained models is consistent with that of the all-atom force field for all lipidations and reproduces key dynamical and structural features of lipid-anchored peptides, such as the solvent-accessible surface area, bilayer penetration depth, and representative conformations of the anchors. The parameters are also validated in simulations of the lipid-anchored peripheral membrane proteins Rheb and Arf1, after comparison with independent all-atom simulations. The parameters, along with mapping schemes for the popular martinize2 tool, are available for download at 10.5281/zenodo.7849262 and also as supporting information.

Martini 3 Coarse-Grained Force Field for Cholesterol.

Cholesterol plays a crucial role in biomembranes by regulating various properties, such as fluidity, rigidity, permeability, and organization of lipid bilayers. The latest version of the Martini model, Martini 3, offers significant improvements in interaction balance, molecular packing, and inclusion of new bead types and sizes. However, the release of the new model resulted in the need to reparameterize many core molecules, including cholesterol. Here, we describe the development and validation of a Martini 3 cholesterol model, addressing issues related to its bonded setup, shape, volume, and hydrophobicity. The proposed model mitigates some limitations of its Martini 2 predecessor while maintaining or improving the overall behavior.

Coarse-grained molecular dynamics simulations of lipid-protein interactions in SLC4 proteins.

The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO 3 -, CO 3 2- , Cl - , Na + , K + , NH 3 and H + necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl - /HCO 3 - exchanger) and NBCe1 (Na + -CO 3 2- cotransporter). Previous computational studies of the outward facing (OF) state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed multiple 50 µs coarse-grained molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1 and NDCBE (a Na + -CO 3 2- /Cl - exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), phosphatidylserine (POPS), and sphingomyelin (POSM). The recently resolved inward-facing (IF) state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, POPC, and POSM in the three studied proteins and their potential roles in the SLC4 transport function, conformational transition and protein dimerization were discussed. Statement of significance: The SLC4 protein family is involved in critical physiological processes like pH and blood pressure regulation and maintenance of ion homeostasis. Its members can be found in various tissues. A number of studies suggest possible lipid regulation of the SLC4 function. However, the protein-lipid interactions in the SLC4 family are still poorly understood. Here we make use of long coarse-grained molecular dynamics simulations to assess the protein-lipid interactions in three SLC4 proteins with different transport modes, AE1, NBCe1, and NDCBE. We identify putative lipid binding sites for several lipid types of potential mechanistic importance, discuss them in the framework of the known experimental data and provide a necessary basis for further studies on lipid regulation of SLC4 function.

Mechanistic insights into robust cardiac IKs potassium channel activation by aromatic polyunsaturated fatty acid analogues.

Voltage-gated potassium (KV) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. KV channel mutations lead to disordered cellular excitability. Loss-of-function mutations, for example, result in membrane hyperexcitability, a characteristic of epilepsy and cardiac arrhythmias. Interventions intended to restore KV channel function have strong therapeutic potential in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise a class of KV channel activators with potential applications in the treatment of arrhythmogenic disorders such as long QT syndrome (LQTS). LQTS is caused by a loss-of-function of the cardiac IKs channel - a tetrameric potassium channel complex formed by KV7.1 and associated KCNE1 protein subunits. We have discovered a set of aromatic PUFA analogues that produce robust activation of the cardiac IKs channel, and a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We determine the mechanisms through which tyrosine PUFA analogues exert strong activating effects on the IKs channel by generating modified aromatic head groups designed to probe cation-pi interactions, hydrogen bonding, and ionic interactions. We found that tyrosine PUFA analogues do not activate the IKs channel through cation-pi interactions, but instead do so through a combination of hydrogen bonding and ionic interactions.

An implementation of the Martini coarse-grained force field in OpenMM.

We describe a complete implementation of Martini 2 and Martini 3 in the OpenMM molecular dynamics software package. Martini is a widely used coarse-grained force field with applications in biomolecular simulation, materials, and broader areas of chemistry. It is implemented as a force field but makes extensive use of facilities unique to the GROMACS software, including virtual sites and bonded terms that are not commonly used in standard atomistic force fields. OpenMM is a flexible molecular dynamics package widely used for methods development and is competitive in speed on GPUs with other commonly used packages. OpenMM has facilities to easily implement new force field terms, external forces and fields, and other nonstandard features, which we use to implement all force field terms used in Martini 2 and Martini 3. This allows Martini simulations, starting with GROMACS topology files that are processed by custom scripts, with all the added flexibility of OpenMM. We provide a GitHub repository with test cases, compare accuracy and performance between GROMACS and OpenMM, and discuss the limitations of our implementation in terms of direct comparison with GROMACS. We describe a use case that implements the Modeling Employing Limited Data method to apply experimental constraints in a Martini simulation to efficiently determine the structure of a protein complex. We also discuss issues and a potential solution with the Martini 2 topology for cholesterol.

Endocannabinoids enhance hKV7.1/KCNE1 channel function and shorten the cardiac action potential and QT interval.

BACKGROUND: Genotype-positive patients who suffer from the cardiac channelopathy Long QT Syndrome (LQTS) may display a spectrum of clinical phenotypes, with often unknown causes. Therefore, there is a need to identify factors influencing disease severity to move towards an individualized clinical management of LQTS. One possible factor influencing the disease phenotype is the endocannabinoid system, which has emerged as a modulator of cardiovascular function. In this study, we aim to elucidate whether endocannabinoids target the cardiac voltage-gated potassium channel KV7.1/KCNE1, which is the most frequently mutated ion channel in LQTS. METHODS: We used two-electrode voltage clamp, molecular dynamics simulations and the E4031 drug-induced LQT2 model of ex-vivo guinea pig hearts. FINDINGS: We found a set of endocannabinoids that facilitate channel activation, seen as a shifted voltage-dependence of channel opening and increased overall current amplitude and conductance. We propose that negatively charged endocannabinoids interact with known lipid binding sites at positively charged amino acids on the channel, providing structural insights into why only specific endocannabinoids modulate KV7.1/KCNE1. Using the endocannabinoid ARA-S as a prototype, we show that the effect is not dependent on the KCNE1 subunit or the phosphorylation state of the channel. In guinea pig hearts, ARA-S was found to reverse the E4031-prolonged action potential duration and QT interval. INTERPRETATION: We consider the endocannabinoids as an interesting class of hKV7.1/KCNE1 channel modulators with putative protective effects in LQTS contexts. FUNDING: ERC (No. 850622), Canadian Institutes of Health Research, Canada Research Chairs and Compute Canada, Swedish National Infrastructure for Computing.

Release of nanodiscs from charged nano-droplets in the electrospray ionization revealed by molecular dynamics simulations.

Electrospray ionization (ESI) is essential for application of mass spectrometry in biological systems, as it prevents the analyte being split into fragments. However, due to lack of a clear understanding of the mechanism of ESI, the interpretation of mass spectra is often ambiguous. This is a particular challenge for complex biological systems. Here, we focus on systems that include nanodiscs as membrane environment, which are essential for membrane proteins. We performed microsecond atomistic molecular dynamics simulations to study the release of nanodiscs from highly charged nano-droplets into the gas phase, the late stage of ESI. We observed two distinct major scenarios, highlighting the diversity of morphologies of gaseous product ions. Our simulations are in reasonable agreement with experimental results. Our work provides a detailed atomistic view of the ESI process of a heterogeneous system (lipid nanodisc), which may give insights into the interpretation of mass spectra of all lipid-protein systems.

CryoEM structures of anion exchanger 1 capture multiple states of inward- and outward-facing conformations.

Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.

Computational Insights into the Role of Cholesterol in Inverted Hexagonal Phase Stabilization and Endosomal Drug Release.

Cholesterol is a major component of many lipid-based drug delivery systems, including cationic lipid nanoparticles. Despite its critical role in the drug release stage, the underlying molecular mechanism by which cholesterol assists in endosomal escape remains unclear. An efficient drug release from the endosome requires endosomal disruption. This disruption is believed to involve a lamellar-to-inverted hexagonal (Lα-HII) phase transition upon fusion of the lipid nanoparticle with the endosomal membrane. We used molecular dynamics simulations to study the structural properties of HII systems composed of an anionic lipid distearoyl phosphatidylserine (DSPS), an ionizable cationic lipid (KC2H), and cholesterol for several hydration levels and molar ratios. This system corresponds to the lipid mixtures in the hypothesized HII structure formed upon fusion and is of interest for the rational design of ionizable cationic lipids, including KC2, for an optimal drug release. Simulations suggest a geometry- and symmetry-driven lipid sorting and cholesterol-DSPS co-location around the water cores. Cholesterol preferentially co-locates with negatively charged saturated DSPS lipids at interstitial angles. The observed cholesterol-DSPS co-location results in an overall increase in the DSPS acyl chains' order parameters, which we propose to assist in stabilizing the HII phase by stretching the DSPS acyl chains for filling the voids formed by three adjacent lipid tubules. Furthermore, a systematic increase in the cholesterol concentration increased the lattice plane spacing and the water core radius but decreased the undulations along the lipid tubule axis. We propose that cholesterol and the degree of saturation/polyunsaturation of the lipid acyl chains, and not the lipid charge, are the main contributors in facilitating the Lα-HII phase transition and stabilizing/destabilizing the formed HII phase, whereas the positive charge of the ionizable cationic lipid promotes the LNP-endosomal membrane adhesion and assists in initiating the fusion process at the local contact area. We also propose that the effect of cholesterol on the HII structure and curvature is the main underlying reason for the well-documented HII stabilization and destabilization at low and high molar concentrations of cholesterol, respectively.

Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers.

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.

Curvature-based sorting of eight lipid types in asymmetric buckled plasma membrane models.

Curvature is a fundamental property of biological membranes and has essential roles in cellular function. Bending of membranes can be induced by their lipid and protein compositions, as well as peripheral proteins, such as those that make up the cytoskeleton. An important aspect of membrane function is the grouping of lipid species into microdomains, or rafts, which serve as platforms for specific biochemical processes. The fluid mosaic model of membranes has evolved to recognize the importance of curvature and leaflet asymmetry, and there are efforts toward evaluating their functional roles. This work investigates the effect of curvature on the sorting of lipids in buckled asymmetric bilayers containing eight lipid types, approximating an average mammalian plasma membrane, through coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field. The simulations reveal that 1) leaflet compositional asymmetry can induce curvature asymmetry, 2) lipids are sorted by curvature to different extents, and 3) curvature-based partitioning trends show moderate to strong correlations with lipid molecular volumes and head to tail bead ratios, respectively. The findings provide unique insights into the role of curvature in membrane organization, and the curvature-based sorting trends should be useful references for later investigations and potentially interpreting the functional roles of specific lipids.

The ugly, bad, and good stories of large-scale biomolecular simulations.

Molecular modeling of large biomolecular assemblies exemplifies a disruptive area holding both promises and contentions. Propelled by peta and exascale computing, several simulation methodologies have now matured into user-friendly tools that are successfully employed for modeling viruses, membranous nano-constructs, and key pieces of the genetic machinery. We present three unifying biophysical themes that emanate from some of the most recent multi-million atom simulation endeavors. Despite connecting molecular changes with phenotypic outcomes, the quality measures of these simulations remain questionable. We discuss the existing and upcoming strategies for constructing representative ensembles of large systems, how new computing technologies will boost this area, and make a point that integrative modeling guided by experimental data is the future of biomolecular computations.

A molecular switch controls the impact of cholesterol on a Kir channel.

SignificanceCholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing second-site mutations at 11 positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508, and F1074 in WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward the space vacated by F508, while in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction.